Human Cytomegalovirus (HCMV) is a significant human pathogen that causes morbidity and mortality in patients with a compromised immune system, including AIDS patients and recipients of organ and bone marrow transplants. In addition, HCMV can cross the placenta and infect the fetus resulting in a high incidence of birth defects such as blindness and deafness. The recent increase in drug-resistant HCMV has demonstrated the need for a more complete understanding of the biology of infection with the goal of identifying novel targets for antiviral therapies. Herpesviruses such as HCMV have a unique proteinaceous layer termed the tegument that contains proteins involved in the initiation of infection as well as virus assembly and egress. The long-term goal of our laboratory is to define the functions of the HCMV pp71 tegument protein. This protein localizes to the nucleus immediately upon infection and enhances the activation of viral genes necessary to successfully establish an active infection. In addition, pp71 localizes to cytoplasmic sites of virus assembly at the late stages of infection. Our recent data demonstrate that the pp71 protein has specific signals capable of directing protein trafficking to three distinct subcellular locales, namely the nucleus, mitochondria and the trans-Golgi network. These findings suggest that the pp71 protein may have additional functions during HCMV infection. In addition, we have identified a specific phosphorylation event that regulates nuclear trafficking of the pp71 protein. This finding supports our central hypothesis that pp71 interaction with and/or modification by specific proteins is critical for the differential subcellular targeting of this protein. We will address our hypothesis in this R03 application by (1) identifying viral and cellular proteins that interact with pp71 and (2) mapping the phosphorylation sites of pp71. In our first aim, we will identify viral and cellular proteins that interact with the pp71 protein using mass spectrometric approaches. Clustal analysis of identified proteins will then be performed by the Human Virus Interactome Resource to reveal novel functions of pp71. In specific aim 2, we will map the phosphorylation sites of pp71 protein in both infected cells and the virion using tandem mass spectrometry approaches. Together, these studies characterizing the pp71 interactome and mapping the phosphorylation sites of pp71 will enhance our understanding of the regulation of this important viral protein. The successful completion of these pilot studies will provide critical preliminary data for a future R01 application examining the regulation and function of the HCMV pp71 tegument protein. A greater understanding of the role of the viral tegument proteins during infection may ultimately lead to the identification of novel targets for antiviral therapies. PUBLIC HEALTH RELEVANCE: Human cytomegalovirus (HCMV) is a significant pathogen, causing disease in immunocompromised patients and congenital infections resulting in birth defects. Recently, drug resistant HCMV has emerged, prompting the need to discover new treatments designed to target this virus. This research will analyze the function and regulation of viral proteins involved in establishing infection and assembling new virus particles, and thus may identify novel targets for anti-HCMV therapies.